Genetic Investigations in Turkish Idiopathic Pancreatitis Patients Show Unique Characteristics

Background/Aims: Pancreatitis is one of the leading causes of digestive system-related hospital admissions, and it has a genetic background in a considerable portion of the patients. In this study, we aimed to investigate the genetic risk factors of idiopathic pancreatitis in Turkish patients and the contribution of copy number variations to the pathogenesis. Materials and Methods: Idiopathic pancreatitis is defined as failure to detect risk factors despite comprehensive clinical assessments. Next-generation sequencing and multiple ligand-dependent probe amplification of PRSS1, SPINK1, CTRC, and CFTR were performed. For further genotype–phenotype correlations, patients were also questioned for the age of onset, family history, and pancreatic divisum. Results: A total of 68 idiopathic pancreatitis cases were enrolled. Variants with potential clinical significance of PRSS1 were identified in 13.4%, SPINK1 in 6.3%, CTRC in 4.7%, and CFTR in 26.5% of the patients. No copy number variants were seen in any of these genes. At least 7.4% of the participants had complex genetic etiology involving 2 genes. Conclusions: At least 42.6% of the participants had a potential genetic risk factor. Five novel genetic variants were identified, and distinctive genetic risk factors of Turkish population were shown. The results showed that genetic etiology was frequent in pancreatitis and it was even more prominent in patients with early-onset disease. Considering that genetic risk factors may be informative for decision-making in the treatment options in addition to providing extensive prognostic value and familial genetic consultation; clinicians need to be more eager to offer genetic tests to pancreatitis patients.


INTRODUCTION
Pancreatitis is one of the leading causes of digestive system-related hospital admissions and leads to a significant amount of morbidities, mortalities, and socioeconomic burden. 1 Pancreatitis is interpreted in 3 subgroups named acute pancreatitis (AP), recurrent AP (RAP), and chronic pancreatitis (CP) according to the onset and frequency of the symptoms. 2Even though these subgroups are known as separate clinical entities, some of the previous studies hypothesized that they may be the different stages of a continuum disease spectrum.
The etiology of pancreatitis is diverse.Alcohol and biliary factors are the most common inducers of AP/RAP, whereas chronic alcohol abuse and continuous smoking are the leading causes of CP. 3 Despite the investigations for other most frequent etiological factors including hypertriglyceridemia, hypercalcemia, infections, or obstructive masses, the cause of pancreatitis cannot be determined in 10%-30% of events and they are called "idiopathic pancreatitis." 4It is known that genetic contributions are identified in a considerable portion of those patients with so-called idiopathic pancreatitis.
The majority of the pancr eatit is-as socia ted genes, like PRSS1, SPINK1, and CTRC, encode proteins involved in trypsin-dependent digestion, whereas CFTR variations lead to pancreatitis by disrupting CFTR channel functions. 1Besides these 4 major genes, CPA1, CEL-HYB hybrid allele, an inversion in CTRB1-CTRB2 locus, CLDN2, CASR, and several other genes were recently reported in pancreatitis. 1,5Studies focusing on variants of these genes in pancreatitis showed that genetic risk factors can be found in 30%-60% of idiopathic RAP and 12%-43% of idiopathic CP. 4 Most of the previous studies in the literature only focused on sequence variants and there are only a limited number of studies which revealed that copy number variants were linked to disease in up to 5% Turk J Gastroenterol 2023; 34(12): 1240-1248 of patients. 6,7Therefore, a concern occurred for possible undiagnosed patients.Also, genetic variants were vastly different in distinct regions of the world, and the single paper disclosing the genetic risk factors of pancreatitis in Turkish patients only evaluated the most frequent pathogenic variants. 8,9In this study, we aimed to investigate the genetic risk factors of pancreatitis in the Turkish population and the contribution of copy number variations to the pathogenesis of idiopathic pancreatitis.

MATERIALS AND METHODS Study Design and Participants
The study was approved by Eskişehir Osmangazi University Non-interventional Clinical Researches Ethics Committee (Approval No: 04, Date: August 6, 2019).Each patient provided informed consent.The clinical information of the patients was obtained from the patients themselves and their hospital records.Both pediatric and adult patients were included.The diagnoses of AP, RAP, or CP were determined according to the latest guidelines. 2,10Idiopathic pancreatitis was defined as failure to detect risk factors despite comprehensive clinical assessments.Therefore, patients with biliary pancreatitis, alcohol abuse (≥40 g/ day or ≥300 g/week), heavy smoking (>20 cigarettes/ day), hyperlipidemia, hypercalcemia, trauma, drug use, and autoimmune pancreatitis were excluded. 3,10[13] For further clinical investigations, patients were questioned for the age of onset and family history.The presence of pancreas divisum was assessed.The age of symptom onset was defined as the age at the time when the first abdominal pain consistent with pancreatitis appeared.Positive family history was determined when there was another family member with pancreatitis in the 3-generation pedigree chart.The diagnosis of pancreas divisum was evaluated by magnetic resonance chola ngiop ancre atogr aphy (MRCP) or endoscopic retrograde chola ngiop ancre atogr aphy (ERCP).If these were not available or gave inconclusive results, the patients were excluded from this part of the study.
For genetic testing, next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) of PRSS1, SPINK1, CTRC, and CFTR genes were performed.The NGS variants were classified according to American Collage of Medical Genetics (ACMG) criteria and variants of uncertain significance (VUS), likely pathogenic and pathogenic variants were presented as "variants with potential clinical significance."A common synonym variant of CTRC, c.180C>T (p.G60G), was interpreted separately as it was previously reported to be a risk factor. 14To compare the frequency of this variant with Turkish controls, whole-exome sequencing data from the Intergen Genetic Diagnosis Center were used as a control.The SALSA MLPA Probemix P242 Pancreatitis and SALSA MLPA Probemix P091 CFTR probes were used to determine the copy number variants of these 4 genes.

Statistical Analysis
Statistical analyses were performed on IBM's Statistical Package for the Social Sciences version 21.0 (IBM Corp., Armonk, NY, USA).Summary statistics of categorical variables were shown as mean with standard deviation (SD), median with interquartile range (IQR), or frequency count with percentage.Fisher exact, Yates, or Pearson chisquare test was used to compare categorical variables and between-group differences, a P-value of <.05 was considered as statistically significant.

Main Points
Among the 11 patients with idiopathic pancreatitis with pancreas divisum, 4 (36.4%) had CFTR variants, and  variants of other genes were not detected (Table 4).The CFTR variants were present in 13 (24.5%)patients without pancreas divisum.In addition, 7 (20.0%)patients with no clinically significant variants had pancreas divisum.
Potential genetic risk factors were found in 60.9% of the patients with childhood-onset symptoms and in 33.3% of the patients with adult-onset symptoms.Pathogenic PRSS1 and SPINK1 variants were predominantly present in patients with childhood-onset disease (Figure 2).Potential genetic risk factors were identified in 81.8% of the patients who had a family history and in 36.4% of the patients who did not have one.
The common c.180C>T variant of CTRC was identified in 17 participants (heterozygous in 14, homozygous in 3, allele frequency: 15.6%).The allele frequency of the variant was 9.4% (393/4186) in our in-house exome sequencing data, so the variant was statistically more frequent in idiopathic pancreatitis patients (P = .027).It was also statistically confirmed that the variant was more frequent in idiopathic CP patients compared to the controls (P = .014).The variant was more common in idiopathic CP than idiopathic RAP + AP group, but the P-value was not statistically significant (P = .239)(Table 5).

DISCUSSION
Various frequencies of genetic variants have been found to contribute to the pathogenesis of pancreatitis in studies so far.As well as the ethnic characteristics of the populations, different outcomes were also related to the demographic/clinical characteristics of the participants, which genetic testing methods were used, and which variants the researchers deemed appropriate to report.In this study, PRSS1, SPINK1, CTRC, and CFTR genes were analyzed by NGS and MLPA to detect both sequence and copy number variants in children and adults with idiopathic pancreatitis and pathogenic/likely pathogenic variants and VUS were reported.
PRSS1 Variants PRSS1 is the first gene associated with pancreatitis, and the most common PRSS1 variants, p.R122H and p.N29I, are expected to be penetrant in as high as 90% of the carriers. 15The highest PRSS1 variant frequencies, 31.3% and 30%, were reported in INSPPIRE studies by Giefer et al 15 in 240 and Dike et al 8 in 333 children patients with RAP/CP respectively, and some smaller studies reported that none of their patients with idiopathic CP or RAP had PRSS1 variations. 13PRSS1 variants were defined in 13.4% of our participants, and the most common was p.R122H, consistent with the previous reports.The higher rate of Giefer et al 15 and Dike et al 8 might be attributed to the demographics of their participants since the studies were performed on pediatric patients. 13The PRSS1 variant frequency of our patients whose symptoms started during the pediatric period (26.1%) was comparable to those studies, and our overall PRSS1 variant frequency was similar to large Asian studies planned in both adults and children. 16The highest CNV rate of PRSS1 was reported as 5.1% in a French study of patients with CP, but no CNVs were detected in the present study. 7INK1 Variants SPINK1 was previously stated to be less risky and considered to cause pancreatitis in hardly 1% of pathogenic variant carriers; however, recent Asian studies make this information controversial.16,17 It is stated that pathogenic SPINK1 variants are usually found in patients with other genetic/environmental risk factors; therefore, it is unclear whether pathogenic SPINK1 variants cause all types of pancreatitis by themselves or act like cofactors.18 SPINK1 variants are especially common in Asia, Wang et al 19 and Liu et al 20 reported variant frequencies of 57.3% and 56.2% in their CP patients, respectively.The SPINK1 variant rate and profile of our study have differed from these previous researches.The variant frequency was 6.3% and 2 novel variants, c.162delT (p.N56Mfs*39) and c.181T>C (p.C61R), were defined.Large deletions of SPINK1 were reported in small case reports, but they were not reported in larger studies, and no copy number variant of SPINK1 was identified in this study too.6,7 CTRC Variants Initial studies indicated that CTRC variants were rare causes of pancreatitis, but later, it was found that a common silent variant, c.180C>T, is present in up to 30% of CP patients and may be a risk factor for pancreatitis. Consistent with most of these studies, CTRC variants were present in 4.8%, and c.180C>T was seen in 26.6% of the participants with an allele frequency of 15.6%.There was a statistically significant difference between the allele frequency of it in both idiopathic pancreatitis patients versus controls (P = .027)and idiopathic CP patients versus controls (P = .014). Itwas also shown that it was more frequent in the idiopathic CP than the idiopathic RAP + AP group, but this correlation has not been proven statistically (P = .239).These results were consistent with previous research remarking that c.180C>T variant is associated with CP, and pancreatitis in general.14,22 Nevertheless, the variant being more common in the CP than in the RAP + AP group supported the conclusion of LaRusch et al 14 that this variant might play a role in disease progression from AP/RAP to CP. Ju3 We did not find CNVs of CTRC in our participants either.

CFTR Variants
In addition to cystic fibrosis, pathogenic variants of CFTR are also responsible for non-classic cystic fibrosis phenotypes like congenital vas deferens agenesis, chronic rhinosinusitis, and pancreatitis. 24Unlike variants causing severe impairment in CFTR channel function and resulting in classical cystic fibrosis, milder variants or variants disturbing bicarbonate permeation lead to a higher risk for pancreatitis, and even variants which are not associated with classical cystic fibrosis, like p.R117H or p.L997F, are reported to increase the risk for pancreatitis. 1,23,25ecause of this, interpreting CFTR variants in pancreatitis is challenging and various studies revealed vastly different outcomes. 4,15,16,22In this study, we classified CFTR variants by their potential pathogenicity for pancreatitis instead of classical cystic fibrosis and it was found that 26.5% of our participants had CFTR variants with potential clinical significance.Like previous studies, most patients had milder variants and none had the most common CFTR variant in classical cystic fibrosis, p.F508del. 1,25We also identified 3 novel CFTR variants c.869+2T>C, c.2605A>G; (p.I869V), c.3401C>T; (p.T1134I), but no segregation or functional analyses were available for these.The CNVs of CFTR in pancreatitis were also rarely studied, and an anomaly was once reported by Sofia et al 5 in 1 of their 80 CP patients.None of our patients had CNVs of CFTR.

Complex Genetic Etiology
Pancreatitis is a multifactorial disease with complex genetic etiology, and most studies revealed that a portion of patients had multiple genetic risk factors.Similar to previous studies, complex genetic etiology was present in at least 5.9% of patients; 2 PRSS1, 1 SPINK1, and 2 CTRC patients also had CFTR variants. 7Previous studies reported a possible synergist effect of SPINK1 and CFTR variants on the development of CP. 26 The majority of our CFTR patients did not have SPINK1 variants and vice versa, but we had a very limited number of patients and it was not possible to come up with any conclusion on this matter.

Age of Symptom Onset, Family History, and Pancreas Divisum
Just as on many other disorders, we found that early-onset pancreatitis was associated with the presence of genetic etiology.In our study, 60.9% of patients with childhoodonset and 33.3% with adult-onset disease had potential genetic risk factors.The majority of patients with pathogenic PRSS1 and SPINK1 variants had the childhoodonset disease, only patient #8 with a highly penetrant p.R122H variant of PRSS1 realized his first symptoms at 71.But since the feeling of pain or suspecting of a serious disease during an abdominal pain attack is such subjective issues, the patient may have earlier disease onset or might have had subclinical markers before.As expected, patients with complex genetic risk factors had earlier disease onset, but interestingly, most patients with pathogenic CFTR variants developed their symptoms after the fifth decade.Two patients with the late-onset disease (patients #6 and #64) had variants (c.3140-26A>G, c.3154T>G) responsive to CFTR modulator therapies, and considering this, clinicians should keep in mind that the genetic testing would also be informative for treatment options of patients with late-onset disease. 27,28e presence of potential genetic risk factors in 81.8% of patients with a family history and 36.4% of others emphasizes that genetic testing should not be limited to patients with a family history.Unfortunately, segregation analysis could not be performed for each patient carrying genetic variants; thus, we do not know if negative family histories on patients with genetic risk factors are caused by reduced penetrance or de novo mutations.
There are conflicting opinions on the association between pancreas divisum and the development of pancreatitis.Although some studies show pancreas divisum is an These results hint at a possible association, but since we had a small group of patients, we could not statistically confirm the link between pancreas divisum and CFTR.
In conclusion, in this study of genetic risk factors in Turkish idiopathic pancreatitis patients, we found that at least 42.6% of our participants had potential genetic risk factors.c.365G>A; p.(R122H) variant of PRSS1 was the most common variant identified in this study, especially in idiopathic CP patients with early-onset disease, and in general, CFTR variants were most frequent.No copy number variations were identified, and 5 single-nucleotide variants were novel (SPINK1: c.162delT, c.181T>C; CFTR: c.869+2T>C, c.2605A>G, c.3401C>T).We also found that the common c.180C>T variant of CTRC was significantly associated with pancreatitis.The biggest limitation of this study was that our participants were a relatively small group of patients, partly due to the COVID-19 pandemic, and segregation analyses could not be completed mainly because of the same reason.Another limitation is that we only performed genetic tests in 4 genes associated with pancreatitis, but other recently described pancreatitis-related genes were not analyzed, so some risk factors in these genes could have been missed.In this study, distinctive genetic risk factors of Turkish pancreatitis patients were shown but larger and long-term studies are still needed to search for other unique genetic variants, investigate pancreas divisum/pancreatitis genetics and understand clinical characteristics of patients with genetic risk factors.

Figure 1 .
Figure 1.The percentages and classifications of identified variants in each gene and total.
Turk J Gastroenterol 2023; 34(12): 1240-1248 Table 3. Characteristics and Classifications of the Identified Variants with Respect to the Clinical Features of the Patients Variant MAF

Two
SPINK1 variants were known pathogenic variants, the novel frameshift in patient #38, c.162delT p.(N56Mfs*39), was classified as likely pathogenic, and the novel missense variant in patient #33, c.181T>C p.(C61R), was classified as VUS.Both patients did not have a family history of pancreatitis, and segregation analysis could not be performed.All 3 CTRC patients had the same variant, but only 1 with very early-onset disease, patient #31, had the variant in the homozygous state, besides a CFTR variant.The 35-year-old asymptomatic parents of this patient were heterozygous, and the CFTR variant was inherited from the mother.Family history was positive in the other 2, genetic testing was performed on the siblings of patient #25, and both of his symptomatic 51-year-old brother and asymptomatic 41-year-old sister had the same variant.

Figure 2 .
Figure 2. Variant frequency by the age of symptom onset.(A) Variant frequency in patients with childhood onset.(B) Variant frequency in patients with adulthood onset.LP, likely pathogenic; P, pathogenic; VUS, variant of uncertain significance.

Table 1 .
Clinical and Demographic Characteristics of the Study Group

Table 4 .
Variant Frequencies in Patients with/Without Pancreas Divisum